In certain applications, such as single molecule DNA sequencing or the evaluation of polymerases, it is necessary to wash labeled biomolecules across a surface. This process inevitably results in the nonspecific binding of labeled molecules to the surface and a concominant increased background fluorescence and false-positive features. Many surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but do little to directly inhibit or suppress the effects of the nonspecific binding of fluorescently labeled molecules. The corresponding increase in background fluorescence as labeled molecules are washed across the surface, combined with the limited fluorescent intensity and lifetime of any single fluorophore, imposes restrictions on the overall imaging capabilities of any single molecule surface chemistry. The spatial resolution of an optical system is limited by the Rayleigh criterion: dR=0.61λ/N.A., where λ is the wavelength of collected photons and N.A. is the numerical aperture of the system. As a result of these optical limitations, current methods for the surface deposition and visualization of fluorescently-labeled single molecules suffer from a number of fundamental limitations. Poisson statistics reveal that a certain fraction of all randomly distributed molecules on a surface will be located within a diffraction limit distance of at least one other molecule, resulting in neither of the two molecules being easily resolvable. As a result, for a given optical setup, as the number of deposited molecules increases, the total number of resolvable molecules reaches a maximum and then decreases. Recent a posteriori methods have been developed using centroid localization or photobleaching to resolve multiple single molecules within a diffraction-limited area with high precision. While some of these techniques could conceivably be used to increase the maximum number of resolvable molecules while using random deposition, they would require precise observation of every photobleaching event to realize a significant degree of accuracy. Given the sensitivity and capture rate limitations of current CCD technology, it would likely not be practical to use these methods to completely resolve a highly dense surface array of single molecules.
Common surface attachment chemistries, for both single molecule and bulk sample surface immobilization, typically involve specific ligand binding, specific covalent coupling, or nonspecific chemiabsorption or physiabsorption. Some examples include biotinstreptavidin or digoxygenin-antidigoxygenin coupling, azide-alkyne cycloaddition coupling (24), coupling between an amine-reactive substrate (eg. aromatic isothiocyanate) and a chemically modified aliphatic 1° amine biomolecule, or absorption to a positively charged poly-electrolyte surface followed by chemical or photochemical crosslinking. Many of these surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but some of them do little to inhibit the effects of nonspecific binding. However, the corresponding increase in background fluorescence as successive fluorescently labeled molecules are washed across the surface limits the overall imaging capabilities of any surface chemistry.
Various other chemical techniques have been developed to minimize background noise while optically imaging single molecules immobilized on a surface. These methods include using fluorescence resonance energy transfer (FRET) to resolve the relative proximity of molecules beyond the diffraction limit, building a negatively charged surface out of a polyelectrolyte multilayer to reduce nonspecific binding of fluorescently labeled nucleotides, using photo-cleavable or chemically cleavable fluorescent labels and extensive washing, and the use of a “smart” hydropolymer shield capable of preventing small molecule binding.
However, there remains a need in the art for a method of resolving single molecules on a surface, especially when random deposition of the molecules is desired.